interferometric scattering microscopy

The principle of interference plays a central role in many imaging methods, including bright-field imaging because it can be described as the interference between the illumination field and the one that has interacted with the object, i.e. through extinction. In fact, even microscopy based on the interference with an external light field is more than one hundred years old.

The first iSCAT-type of measurements were performed in the biophysics community in the 1990s.{{Cite journal|last1=AMOS|first1=L. A.|last2=AMOS|first2=W. B.|date=1991-01-01|title=The bending of sliding microtubules imaged by confocal light microscopy and negative stain electron microscopy|journal=Journal of Cell Science|volume=1991|issue=Supplement 14|pages=95–101|doi=10.1242/jcs.1991.supplement_14.20|pmid=1715872|pmc=2561856|issn=0021-9533|doi-access=free}} A systematic development of the method for the detection of nano-objects started in the early 2000s as a general effort to explore fluorescence-free options for studying single molecules and nano-objects.{{Cite journal|last1=Lindfors|first1=K.|last2=Kalkbrenner|first2=T.|last3=Stoller|first3=P.|last4=Sandoghdar|first4=V.|date=July 2004|title=Detection and Spectroscopy of Gold Nanoparticles Using Supercontinuum White Light Confocal Microscopy|journal=Physical Review Letters|volume=93|issue=3|pages=037401|doi=10.1103/physrevlett.93.037401|pmid=15323866|bibcode=2004PhRvL..93c7401L|issn=0031-9007}} In particular, gold nanoparticles down to a size of 5 nm were imaged via the interference of their scattered light with a reflected beam from the cover-slip supporting them. Using a supercontinuum laser additionally allowed for recording the particles' plasmon spectra. The early measurements were limited by residual speckle-like background. A new approach to background subtraction and the acronym iSCAT were introduced in 2009.{{Cite journal|last1=Kukura|first1=Philipp|last2=Ewers|first2=Helge|last3=Müller|first3=Christian|last4=Renn|first4=Alois|last5=Helenius|first5=Ari|last6=Sandoghdar|first6=Vahid|date=2009-11-01|title=High-speed nanoscopic tracking of the position and orientation of a single virus|journal=Nature Methods|volume=6|issue=12|pages=923–927|doi=10.1038/nmeth.1395|pmid=19881510|s2cid=10615639|issn=1548-7091}} Since then, a series of important works has been reported by various groups.{{Cite journal|last=Hsieh|first=Chia-Lung|date=September 2018|title=Label-free, ultrasensitive, ultrahigh-speed scattering-based interferometric imaging|journal=Optics Communications|volume=422|pages=69–74|doi=10.1016/j.optcom.2018.02.058|bibcode=2018OptCo.422...69H|s2cid=125505926 |issn=0030-4018}}{{Cite book|title=Label-free super-resolution microscopy|others=Astratov, Vasily|date = 31 August 2019|isbn=978-3-030-21722-8|location=Cham|oclc=1119720519}}{{Cite journal|last1=Young|first1=Gavin|last2=Kukura|first2=Philipp|date=2019-06-14|title=Interferometric Scattering Microscopy|journal=Annual Review of Physical Chemistry|volume=70|issue=1|pages=301–322|doi=10.1146/annurev-physchem-050317-021247|pmid=30978297|bibcode=2019ARPC...70..301Y|s2cid=195661687 |issn=0066-426X}}{{Cite journal|last1=Taylor|first1=Richard W.|last2=Sandoghdar|first2=Vahid|date=2019-07-17|title=Interferometric Scattering Microscopy: Seeing Single Nanoparticles and Molecules via Rayleigh Scattering|journal=Nano Letters|volume=19|issue=8|pages=4827–4835|doi=10.1021/acs.nanolett.9b01822|pmid=31314539|pmc=6750867|bibcode=2019NanoL..19.4827T|issn=1530-6984|doi-access=free}} Notably, further innovations in background and noise suppression have led to the development of new quantification methods such as mass photometry (originally introduced as iSCAMS), in which ultrasensitive and accurate interferometric detection is converted into a quantitative means for measuring the molecular mass of single biomolecules.{{Cite journal|last1=Young|first1=Gavin|last2=Hundt|first2=Nikolas|last3=Cole|first3=Daniel|last4=Fineberg|first4=Adam|last5=Andrecka|first5=Joanna|last6=Tyler|first6=Andrew|last7=Olerinyova|first7=Anna|last8=Ansari|first8=Ayla|last9=Marklund|first9=Erik G.|last10=Collier|first10=Miranda P.|last11=Chandler|first11=Shane A.|date=2018-04-27|title=Quantitative mass imaging of single biological macromolecules|journal=Science|language=en|volume=360|issue=6387|pages=423–427|doi=10.1126/science.aar5839|issn=0036-8075|pmc=6103225|pmid=29700264|bibcode=2018Sci...360..423Y}} {{clear}}

When a reference light is superposed with an object's scattered light, the intensity at the detector can be described by,

$I\_\{det\}\; \backslash propto\; |\backslash overline\{E\_r\}\; +\; \backslash overline\{E\_s\}|^2\; =\; I\_r\; +\; I\_s\; +\; 2\; E\_r\; E\_s\; \backslash cos\; \backslash phi$

where $\backslash overline\{E\_r\}\; =\; E\_r\; e^\{i\; \backslash phi\_r\}$ and $\backslash overline\{E\_s\}\; =\; E\_s\; e^\{i\; \backslash phi\_s\}$ are the complex electric fields of the reference and scattered light. The resulting terms are the intensity of the reference beam $(I\_r\; =\; |\backslash overline\{E\_r\}|^2)$, the pure scattered light from the object $(I\_s\; =\; |\backslash overline\{E\_s\}|^2)$, and the cross-term $(2\; E\_r\; E\_s\; \backslash cos\; \backslash phi)$ which contains a phase $\backslash phi\; =\; \backslash phi\_r\; -\; \backslash phi\_s$. This phase comprises a Gouy phase component from the variations of the wave vectors, a scattering phase component from the material properties of the object, and a sinusoidally modulating phase component which depends on the position of the particle.

In general, the reference beam can take a different path than the scattered light within the optical setup, as long as they are coherent and interfere on the detector. However, the technique becomes simpler and more stable if both beams share the same optical path. Therefore, the reflected light off the cover-slip or the transmitted beam through the sample is typically used as the reference. For the interference to occur, it is necessary that both light waves (scattered light and reference light) are coherent. Interestingly, a light source with a large coherence length on the order of meters or more (like in modern narrow-band laser systems) is typically not needed. In the most common iSCAT realization schemes where the reflected light of a cover-slip is used as a reference and the scattering particle is not more than a few hundreds of nanometers above the glass, even "incoherent" light, e.g. from LEDs, can be used.{{Cite journal|last1=Daaboul|first1=G.G.|last2=Vedula|first2=R.S.|last3=Ahn|first3=S.|last4=Lopez|first4=C.A.|last5=Reddington|first5=A.|last6=Ozkumur|first6=E.|last7=Ünlü|first7=M.S.|date=January 2011|title=LED-based Interferometric Reflectance Imaging Sensor for quantitative dynamic monitoring of biomolecular interactions|journal=Biosensors and Bioelectronics|volume=26|issue=5|pages=2221–2227|doi=10.1016/j.bios.2010.09.038|pmid=20980139|issn=0956-5663}}

iSCAT has been used in multiple applications. These can be grouped roughly as:

• Microtubules{{Cite journal|last1=Vala|first1=Milan|last2=Bujak|first2=Łukasz|last3=García Marín|first3=Antonio|last4=Holanová|first4=Kristýna|last5=Henrichs|first5=Verena|last6=Braun|first6=Marcus|last7=Lánský|first7=Zdeněk|last8=Piliarik|first8=Marek|date=2021-01-25|title=Nanoscopic Structural Fluctuations of Disassembling Microtubules Revealed by Label-Free Super-Resolution Microscopy|journal=Small Methods|language=en|volume=5|issue=4|pages=2000985|doi=10.1002/smtd.202000985|pmid=34927839 |s2cid=234070923 |issn=2366-9608|doi-access=free}}
• Live-cell intracellular organelles{{cite journal |last1=Küppers |first1=Michelle |last2=Albrecht |first2=David |last3=Kashkanova |first3=Anna D. |last4=Lühr |first4=Jennifer |last5=Sandoghdar |first5=Vahid |title=Confocal interferometric scattering microscopy reveals 3D nanoscopic structure and dynamics in live cells |journal=Nature Communications |date=7 April 2023 |volume=14 |issue=1 |pages=1962 |doi=10.1038/s41467-023-37497-7 |pmid=37029107 |language=en |issn=2041-1723|pmc=10081331 |bibcode=2023NatCo..14.1962K }}
• Lipid nano/microdomains{{Cite journal|last1=de Wit|first1=Gabrielle|last2=Danial|first2=John S. H.|last3=Kukura|first3=Philipp|last4=Wallace|first4=Mark I.|date=2015-09-23|title=Dynamic label-free imaging of lipid nanodomains|journal=Proceedings of the National Academy of Sciences|volume=112|issue=40|pages=12299–12303|doi=10.1073/pnas.1508483112|pmid=26401022|pmc=4603517|bibcode=2015PNAS..11212299D|issn=0027-8424|doi-access=free}}
• Single virus assembly{{Cite journal|title=Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome|last1=Garmann|first1=Rees F.|last2=Goldfain|first2=Aaron M.|last3=Manoharan|first3=Vinothan N.|journal=Proceedings of the National Academy of Sciences |year=2018|volume=116 |issue=45 |pages=22485–22490 |doi=10.1073/pnas.1909223116 |pmid=31570619 |pmc=6842639 |arxiv=1802.05211|doi-access=free }}
• Time-dependent iSCAT (StroboSCAT){{Cite journal|last1=Penwell|first1=Samuel B.|last2=Ginsberg|first2=Lucas D. S.|last3=Noriega|first3=Rodrigo|last4=Ginsberg|first4=Naomi S.|date=2017-09-18|title=Resolving ultrafast exciton migration in organic solids at the nanoscale|journal=Nature Materials|volume=16|issue=11|pages=1136–1141|doi=10.1038/nmat4975|pmid=28920937|issn=1476-1122|arxiv=1706.08460|bibcode=2017NatMa..16.1136P|s2cid=205416059}}

• Single virus tracking in vitro
• Single virus tracking during early-stage infection in cells{{Cite journal|last1=Huang|first1=Yi-Fan|last2=Zhuo|first2=Guan-Yu|last3=Chou|first3=Chun-Yu|last4=Lin|first4=Cheng-Hao|last5=Chang|first5=Wen|last6=Hsieh|first6=Chia-Lung|date=2017-01-13|title=Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells|journal=ACS Nano|volume=11|issue=3|pages=2575–2585|doi=10.1021/acsnano.6b05601|pmid=28067508|issn=1936-0851}}
• Microsecond single particle tracking on a living cell membrane{{Cite journal|last1=Taylor|first1=Richard W.|last2=Mahmoodabadi|first2=Reza Gholami|last3=Rauschenberger|first3=Verena|last4=Giessl|first4=Andreas|last5=Schambony|first5=Alexandra|last6=Sandoghdar|first6=Vahid|date=July 2019|title=Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane|url=https://www.nature.com/articles/s41566-019-0414-6|journal=Nature Photonics|language=en|volume=13|issue=7|pages=480–487|doi=10.1038/s41566-019-0414-6|bibcode=2019NaPho..13..480T|s2cid=195094855|issn=1749-4893}}
• Motor protein tracking{{Citation|last1=Andrecka|first1=J.|title=Interferometric Scattering Microscopy for the Study of Molecular Motors|date=2016|work=Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods|pages=517–539|publisher=Elsevier|isbn=978-0-12-809267-5|last2=Takagi|first2=Y.|last3=Mickolajczyk|first3=K.J.|last4=Lippert|first4=L.G.|last5=Sellers|first5=J.R.|last6=Hancock|first6=W.O.|last7=Goldman|first7=Y.E.|last8=Kukura|first8=P.|volume=581|doi=10.1016/bs.mie.2016.08.016|pmid=27793291|pmc=5098560}}{{Cite journal|last1=Bujak|first1=Łukasz|last2=Holanová|first2=Kristýna|last3=García Marín|first3=Antonio|last4=Henrichs|first4=Verena|last5=Barvík|first5=Ivan|last6=Braun|first6=Marcus|last7=Lánský|first7=Zdeněk|last8=Piliarik|first8=Marek|date=2021-08-16|title=Fast Leaps between Millisecond Confinements Govern Ase1 Diffusion along Microtubules|url=https://onlinelibrary.wiley.com/doi/10.1002/smtd.202100370|journal=Small Methods|language=en|volume=5|issue=10|pages=2100370|doi=10.1002/smtd.202100370|pmid=34927934 |s2cid=238695264 |issn=2366-9608}}

• Single molecule detection by absorption{{Cite journal|last1=Kukura|first1=Philipp|last2=Celebrano|first2=Michele|last3=Renn|first3=Alois|last4=Sandoghdar|first4=Vahid|date=2010-11-11|title=Single-Molecule Sensitivity in Optical Absorption at Room Temperature|journal=The Journal of Physical Chemistry Letters|volume=1|issue=23|pages=3323–3327|doi=10.1021/jz101426x|issn=1948-7185}}
• Single protein sensing{{Cite journal|last1=Piliarik|first1=Marek|last2=Sandoghdar|first2=Vahid|date=2014-07-29|title=Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites|journal=Nature Communications|volume=5|issue=1|pages=4495|doi=10.1038/ncomms5495|pmid=25072241|arxiv=1310.7460|bibcode=2014NatCo...5.4495P|issn=2041-1723|doi-access=free}}{{Cite journal|last1= Dahmardeh |first1= Mahyar|last2= Mirzaalian Dastjerdi|first2=Houman|last3=Mazal|first3=Hisham|last4=Köstler|first4=Harald|last5=Sandoghdar|first5=Vahid|date=2023-02-27|title= Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa|journal=Nature Methods|volume=20|issue=3|pages=442–447|doi=10.1038/s41592-023-01778-2|pmid=36849549|pmc= 9998267|issn=1548-7105|doi-access=free}}
• Single protein tracking{{Cite journal|last1=Spillane|first1=Katelyn M.|last2=Ortega-Arroyo|first2=Jaime|last3=de Wit|first3=Gabrielle|last4=Eggeling|first4=Christian|last5=Ewers|first5=Helge|last6=Wallace|first6=Mark I.|last7=Kukura|first7=Philipp|date=2014-08-27|title=High-Speed Single-Particle Tracking of GM1 in Model Membranes Reveals Anomalous Diffusion due to Interleaflet Coupling and Molecular Pinning|journal=Nano Letters|volume=14|issue=9|pages=5390–5397|doi=10.1021/nl502536u|pmid=25133992|pmc=4160260|bibcode=2014NanoL..14.5390S|issn=1530-6984|doi-access=free}}
• Mass photometry

• Single ions entering/leaving battery cathode{{Cite web|last=Lavars|first=Nick|date=June 24, 2021|title=Real-time view of lithium ions in motion opens door to next-gen batteries|url=https://newatlas.com/energy/real-time-view-lithium-ions-next-gen-batteries/|access-date=2021-06-24|website=New Atlas|language=en-US}}

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Category:Microscopy